Cloning and expression of type XII collagen isoforms during bovine adipogenesis

In order to isolate candidate genes involved in bovine adipocyte differentiation, we have constructed a subtraction library from a clonal bovine intra-muscular pre-adipocyte (BIP) cell line using the suppression subtractive hybridization method. We have isolated a set of subtracted cDNA fragments whose respective mRNA levels are up-regulated during the adipogenic differentiation of BIP cells, and cloned cDNAs from a differentiated BIP-lambda ZAP II cDNA library. Two cDNA clones were highly homologous to the sequence of mouse and human type XII collagen alpha-1, determined by a BLAST homology search. As type XII collagen has been reported to have four types of splicing isoform, two clones were determined to be XII-1 and XII-2 splicing isoforms, respectively, because of a difference in the C-terminal NC1 domain. From the expression analysis of type XII collagen, the XIIA-2 isoform was mainly expressed in differentiated BIP cells and adipose tissues. Although the function of type XII collagen has not been established as yet, these results suggest that type XII collagen may be associated with adipocyte differentiation and adipose formation in cattle and is a potentially useful marker for adipogenesis.     

Gene expression patterns of bovine perimuscular preadipocytes during adipogenesis

Bovine perimuscular fat (PMF) preadipocytes were induced to undergo adipogenesis in vitro in our recent study to define the expression patterns of genes involved in the differentiation process. Based on the understanding of the interaction among adipogenic genes, a broad overview of gene expression profile in the differentiating PMF preadipocytes was evaluated using bovine specific DNA microarray from day 2 to 8 post-differentiation induction. A total of 100 significantly differentially expressed genes were detected between differentiated and control cells including those involved in several biochemical pathways and cellular/molecular signaling. In addition, quantitative real-time PCR validated that typical adipogenic genes were up-regulated at early differentiation in the preadipocytes. These results suggest that the PMF preadipocyte system is available as a novel in vitro model for molecular adipogenesis studies in the bovine and that a series of genes are switched on/off during early events associated with adipogenesis.     

Catalase is required for peroxisome maintenance during adipogenesis

Although obesity contributes to the onset and pathogenesis of metabolic diseases, it has been repeatedly demonstrated that being overweight or mildly obese carries a survival advantage compared with being thin or normal-weight. This relationship is called the obesity paradox. Hence, it is necessary to clarify the underlying mechanism of obesity onset for the prevention and treatment of these diseases. Catalase is distributed in peroxisomes under normal redox conditions and catalase activity is increased during the differentiation of 3T3-L1 preadipocytes to adipocytes. Although peroxisomes are responsible for lipid metabolism, the role of peroxisomal catalase in the process of lipid accumulation remains unclear. The present study aimed to investigate the relationships among catalase activity, peroxisome content, and lipid accumulation during the differentiation of 3T3-L1 preadipocytes to adipocytes. Increased catalase activity and lipid accumulation were observed during the differentiation of preadipocytes. Silencing of catalase by small interfering RNA or treatment with 3-amino-1,2,4-triazole (3-AT), a catalase inhibitor, resulted in reduced lipid accumulation. Inhibition of catalase activity in peroxisomes increases hydrogen peroxide (H₂O₂) levels, which results in a reduction of peroxisome content. Extracellular H₂O₂ had no influence on lipid accumulation during differentiation. The occurrence of autophagy was clearly enhanced in cells treated with 3-AT. Spautin-1, an inhibitor of autophagy flux, protected against a reduction in lipid accumulation by treatment with 3-AT. Our data provide evidence that catalase protects against the degradation of peroxisomes via the occurrence of autophagy triggered by the generation of H₂O₂ in peroxisomes. These results suggest that catalase in peroxisomes is crucial to adipogenesis.     

Type I collagen reduces lipid accumulation during adipogenesis of preadipocytes 3T3-L1 via the YAP-mTOR-autophagy axis

The extracellular matrix (ECM) regulates cell behavior through signal transduction and provides a suitable place for cell survival. As one of the major components of the extracellular matrix, type I collagen is involved in regulating cell migration, proliferation and differentiation. We present a system in which 3T3-L1 preadipocyte cells are induced for adipogenic differentiation on type I collagen coated dishes. Our previous study has found that type I collagen inhibits adipogenic differentiation via YAP activation. Here we further reveal that type I collagen inactivates autophagy by up-regulating mTOR activity via the YAP pathway. Under collagen-coating conditions, co-localization of lysosomes with mTOR was increased and the level of downstream protein p-S6K was elevated, accompanied by a decrease in the level of autophagy. Autophagy is negatively correlated with adipogenesis under type I collagen coating. Through the YAP-autophagy axis, type I collagen improves glycolipid metabolism accompanied by increased mitochondrial content, enhanced glucose uptake, reduced release of free fatty acids (FFAs) and decreased intracellular lipid accumulation. Our findings provide insight into the strategy for dealing with obesity: Type I collagen or the drugs with inhibitory effects on autophagy or YAP, have a potential to accelerate the energy metabolism of adipose tissue, so as to better maintain the homeostasis of glucose and lipids in the body, which can be used for achieving weight loss.     

Secreted cathepsin L generates endostatin from collagen XVIII

Endostatin, an inhibitor of angiogenesis and tumor growth, was identified originally in conditioned media of murine hemangioendothelioma (EOMA) cells. N-terminal amino acid sequencing demonstrated that it corresponds to a fragment of basement membrane collagen XVIII. Here we report that cathepsin L is secreted by EOMA cells and is responsible for the generation of endostatin with the predicted N-terminus, while metalloproteases produce larger fragments in a parallel processing pathway. Efficient endostatin generation requires a moderately acidic pH similar to the pericellular milieu of tumors. The secretion of cathepsin L by a tumor cell line of endothelial origin suggests that this cathepsin may play a role in angiogenesis. We propose that cleavage within collagen XVIII's protease-sensitive region evolved to regulate excessive proteolysis in conditions of induced angiogenesis.     

Cell shape alteration during adipogenesis is associated with coordinated matrix cues

Obesity has become one of the leading pathophysiologic disorders in recent years. Adipose tissue is the main tissue related to obesity and is known to play a role in various physiological complications, including type 2 diabetes. To better understand how the fat tissue develops, we used an in vitro live cell imaging system to quantify the adipogenesis by means of nondestructive digital imaging to monitor the accumulation of intracellular lipid droplets (LDs), a hallmark of adipogenesis, from the macro- to the micro-scale. Analyzing the cells’ shape at the single-cell level allows to quantify the cells’ shape change from a fibroblast to spherical morphology, indicating the start of adipogenesis. To reveal the molecular alterations, we applied a proteomic approach using high-resolution mass spectrometry of the proliferation, confluent fibroblasts and of adipocytes. During this process, we noted the reorganization of the cells’ extracellular matrix (ECM) network microenvironment from fibrillary collagen types I, III and V to collagens IV and VI, which affected the cells niche. The changes in ECM are translated for cytoskeleton remodeling according to cell fate-determining mechanisms. We quantified the cytoskeleton rearrangement of long oriented actin fibers or short cortical and disorganized fibers, associated with LDs accumulation in adipocytes. Developing in vitro models and analytical methods enable us to study differentiation into adipocytes that will advance our understanding regarding the niche conditions that affect adipogenesis. Consequently, this will enable the development of new modalities to prevent obesity and its deleterious outcomes and to develop potential treatments to battle pathophysiology-related diseases.     

The collagen type XVIII endostatin domain is co-localized with perlecan in basement membranes in vivo.

The C-terminal globular endostatin domain of collagen type XVIII is anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. In contrast, many of its cell biological properties are still unknown. We systematically localized the mRNA of collagen type XVIII with the help of in situ hybridization (ISH) and detected it in epithelial and mesenchymal cells of almost all organ systems throughout mouse development. Light and electron microscopic immunohistochemistry (IHC) revealed that the endostatin domain is a widespread component of almost all epithelial basement membranes in all major developing organs, and in all basement membranes of capillaries and blood vessels. Furthermore, quantitative immunogold double labeling demonstrated a co-localization of 50% of the detected endostatin domain together with perlecan in basement membranes in vivo. We conclude that the endostatin domain of collagen type XVIII plays a role, even in early stages of mouse development, other than regulating angiogenesis. In the adult, the endostatin domain could well be involved in connecting collagen type XVIII to the basement membrane scaffolds. At least in part, perlecan appears to be an adaptor molecule for the endostatin domain in basement membranes in vivo.     

Drosophila type XV/XVIII collagen, Mp, is involved in Wingless distribution

Multiplexin (Mp) is the Drosophila orthologue of vertebrate collagens XV and XVIII. Like them, Mp is widely distributed in the basement membranes of the developing embryos, including those of neuroblasts in the central and peripheral nervous systems, visceral muscles of the gut, and contractile cardioblasts. Here we report the identification of mutant larvae bearing piggyBac transposon insertions that exhibit decrease Mp production associated with abdominal cuticular and wing margin defects, malformation of sensory organs and impaired sensitivity to physical stimuli. Additional findings include the abnormal ultrastructure of fatbody associated with abnormal collagen IV deposition, and reduced Wingless deposition. Collectively, these findings are consistent with the notion that Mp is required for the proper formation and/or maintenance of basement membrane, and that Mp may be involved in establishing the Wingless signaling gradients in the Drosophila embryo.     

NNMT is induced dynamically during beige adipogenesis in adipose tissues depot-specific manner

Journal of Physiology and Biochemistry - Nicotinamide N-methyltransferase (NNMT) is a novel regulator, shown recently to regulate adipose tissue energy expenditure partly through changing...     

Lack of collagen XVIII/endostatin results in eye abnormalities

Mice lacking collagen XVIII and its proteolytically derived product endostatin show delayed regression of blood vessels in the vitreous along the surface of the retina after birth and lack of or abnormal outgrowth of retinal vessels. This suggests that collagen XVIII/endostatin is critical for normal blood vessel formation in the eye. All basement membranes in wild-type eyes, except Descemet's membrane, showed immunogold labeling with antibodies against collagen XVIII. Labeling at sites where collagen fibrils in the vitreous are connected with the inner limiting membrane and separation of the vitreal matrix from the inner limiting membrane in mutant mice indicate that collagen XVIII is important for anchoring vitreal collagen fibrils to the inner limiting membrane. The findings provide an explanation for high myopia, vitreoretinal degeneration and retinal detachment seen in patients with Knobloch syndrome caused by loss-of-function mutations in collagen XVIII.     

Type V collagen regulates the assembly of collagen fibrils in cultures of bovine vascular smooth muscle cells

Vascular smooth muscle cells (SMCs), the major cellular constituent of the medial layer of an artery, synthesize the majority of connective tissue proteins, including fibrillar collagen types I, III, and V/XI. Proper collagen synthesis and deposition, which are important for the integrity of the arterial wall, require the antioxidant vitamin C. Vitamin C serves as cofactor for the enzymes prolyl and lysyl hydroxylase, which are responsible for the proper hydroxylation of collagen. Here, the role of type V collagen in the assembly of collagen fibrils in the extracellular matrix (ECM) of cultured vascular SMCs was investigated. Treatment of SMCs with vitamin C resulted in a dramatic induction in the levels of the cell‐layer associated pepsin‐resistant type V collagen, whereas only a minor induction in the levels of types I and III collagen was detected. Of note, the deposition of type V collagen was accompanied by the formation of striated collagen fibrils in the ECM. Immunohistochemistry demonstrated that type V collagen, but not type I collagen, became masked as collagen fibrils matured. Furthermore, the relative ratio of type V to type I collagen decreased as the ECM matured as a function of days in culture, and this decrease was accompanied by an increase in the diameter of collagen fibrils. Together these results suggest that the masking of type V collagen is caused by its internalization on continuous deposition of type I collagen on the exterior of the fibril. Furthermore, they suggest that type V collagen acts as framework for the initial assembly of collagen molecules into heterotypic fibrils, regulating the diameter and architecture of these fibrils. J. Cell. Biochem. 80:146–155, 2000. © 2000 Wiley‐Liss, Inc.     

Type V collagen regulates the assembly of collagen fibrils in cultures of bovine vascular smooth muscle cells

Vascular smooth muscle cells (SMCs), the major cellular constituent of the medial layer of an artery, synthesize the majority of connective tissue proteins, including fibrillar collagen types I, III, and V/XI. Proper collagen synthesis and deposition, which are important for the integrity of the arterial wall, require the antioxidant vitamin C. Vitamin C serves as cofactor for the enzymes prolyl and lysyl hydroxylase, which are responsible for the proper hydroxylation of collagen. Here, the role of type V collagen in the assembly of collagen fibrils in the extracellular matrix (ECM) of cultured vascular SMCs was investigated. Treatment of SMCs with vitamin C resulted in a dramatic induction in the levels of the cell-layer associated pepsin-resistant type V collagen, whereas only a minor induction in the levels of types I and III collagen was detected. Of note, the deposition of type V collagen was accompanied by the formation of striated collagen fibrils in the ECM. Immunohistochemistry demonstrated that type V collagen, but not type I collagen, became masked as collagen fibrils matured. Furthermore, the relative ratio of type V to type I collagen decreased as the ECM matured as a function of days in culture, and this decrease was accompanied by an increase in the diameter of collagen fibrils. Together these results suggest that the masking of type V collagen is caused by its internalization on continuous deposition of type I collagen on the exterior of the fibril. Furthermore, they suggest that type V collagen acts as framework for the initial assembly of collagen molecules into heterotypic fibrils, regulating the diameter and architecture of these fibrils.     

Type XIV collagen is a variant of undulin.

We have isolated undulin, an extracellular matrix protein associated with the surface of collagen fibrils, from chicken embryos. The protein showed a molecular mass of about 600 kDa and is composed of three 210-kDa subunits linked by reducible as well as non-reducible bonds. In contrast to human undulin which reportedly is devoid of collagenous sequences, the chicken protein contained a short triple-helical segment that was sensitive to digestion by bacterial collagenase. Screening of an expression library with affinity-purified antibodies yielded two cDNA clones specific for chicken undulin. Analysis of the amino acid sequence deduced from the nucleotide sequence of these clones showed that the human and the chicken protein shared 71% sequence identity. At the amino-terminus both polypeptides contained several similar repeats related to the type III modules found in fibronectin. Towards the carboxyl terminus, however, the two sequences diverged substantially from each other. While the human sequence terminated in a proline-rich segment, the chicken sequence continued with a domain related to von Willebrand factor, with a domain similar to the noncollagenous domain NC4 of type IX collagen and with a typical collagenous triple helix. A short segment of this sequence was found to be identical with the published sequence of a bovine peptide derived from type XIV collagen. Our protein must therefore represent chicken type XIV collagen. One way to explain these results is the possibility that undulin exists in at least two alternatively spliced variants, one lacking the collagenous domain, as described initially for human undulin, and one containing the triple-helical domain, as found in type XIV collagen.     

The multiple functions of collagen XVIII in development and disease

Collagen XVIII is a heparan sulphate proteoglycan which is expressed ubiquitously in different basement membranes throughout the body. Its C-terminal fragment, endostatin, has been found to inhibit angiogenesis and tumor growth by restricting endothelial proliferation and migration and inducing apoptosis of endothelial cells. Collagen XVIII has three variants, of which the shortest one is found in most vascular and epithelial BM structures, whereas the longer variants are found especially in the liver. The longest or frizzled variant has a cysteine-rich domain in its N-terminus that has been shown to inhibit Wnt signaling in vitro. The presence of collagen XVIII homologues in organisms such as C. elegans, Xenopus laevis, zebrafish and chick suggests a fundamental role for this BM collagen. Mutations in the collagen XVIII gene lead to the Knobloch syndrome, which is characterized by high myopia, vitreoretinal degeneration with retinal detachment, macular abnormalities and occipital encephalocele. Mice lacking collagen XVIII also show several ocular abnormalities. This suggests that in physiological conditions collagen XVIII is mostly needed for the proper development of the eye. Moreover, it appears to be needed for the structural stability of basement membranes in several other organs, and increasing evidence shows its importance for other organs in non-physiological situations such as atherosclerosis, glomerulonephritis or other type of tissue damage. This review focuses on clarifying the roles of collagen XVIII and its variants and domains in various physiological and pathological conditions.